Detection of Mitotic Neuroblasts Provides Additional Evidence of Steady-State Neurogenesis in the Adult Small Intestinal Myenteric Plexus

Binucleated ganglionic cells express Hu and PGP9.5. Orthogonal (merged and color-segregated) views of a 3D confocal microscopy image showing that a DAPI-stained- (gray), Hu- (green), and PGP9.5- (red) immunolabeled cell contains two nuclei. XY, YZ, and XY planes of the orthogonal views are denoted for every merged and panel-segregated image. Scale bar, 2 μm. Additional Hu-immunolabeled binucleated cell with conjoined nuclei is shown in Extended Data Figure 2-1.

Evidence of S and G2/M phases of cell cycle in a population of Hu-immunolabeled cells from the small intestinal MP tissue of adult healthy mice. Flow analyses of nuclei isolated from the LM–MP tissue from adult healthy mice housed in a barrier facility at BIDMC when immunolabeled with directly conjugated anti-Hu 488 antibody and costained with a DNA-binding dye NucBlue show that (A) significant proportion of events present with detectable NucBlue-labeling and hence are annotated as nuclei. B, Using a Hu-unstained population of NucBlue-labeled nuclei, we create gates for Hu-immunolabeled cells, which (C) contain Hu-labeled nuclei in a sample that was immunolabeled with a directly conjugated Hu antibody. D, This population of isolated Hu- and NucBlue-labeled nuclei from adult murine small intestinal LM–MP tissues from mice from the BIDMC mouse colony, when analyzed using fluorescence intensity of NucBlue as a marker for DNA content, showed that while 88.0% of nuclei have DNA content (2N) expected in the G1 phase cells, 2.71% of Hu⁺ nuclei contain DNA content that corresponds to cells in S-phase (DNA replication phase), and 7.37% of Hu⁺ nuclei contain DNA content that corresponds to cells in G2/M phase (mitotic phase). The enumeration of nuclei does not include those that show less than 2N DNA content. E, Flow analysis of nuclei isolated from LM–MP of similarly aged mice housed at JHU, which were immunolabeled with anti-Hu 647 antibody postfixation and were stained with the DNA dye DAPI, shows the presence of 90.0% of nuclei have DNA content (2N) expected in the G1 phase cells, 2.61% of Hu⁺ nuclei contain DNA content that corresponds to cells in S-phase (DNA replication phase), and 7.39% of Hu⁺ nuclei contain DNA content that corresponds to cells in G2/M phase (mitotic phase). Flow analyses gates for DAPI-labeled and Hu-immunolabeled nuclei from experiment performed at JHU are shown in Extended Data Figure 3-1.

Cell proliferation marker pH3 is detected in neurons and other cells of the adult healthy MP tissue. Confocal microscopy shows that the (A) LM–MP tissue from an adult healthy mouse that was housed at JHU, when stained with unconjugated antibodies against pH3 (red) and the pan-neuronal marker Hu (green), shows the presence of pH3 immunoreactivity in ganglionic cells that do express Hu (yellow arrow) and those that do not express Hu (cyan arrow). pH3 immunoreactivity is also detected in extraganglionic cells in this tissue (white arrow) that are presumed to be LM cells. B, Magnified image of a myenteric ganglia labeled with antibodies against pH3 (red) and Hu (green) again shows the presence of pH3-immunoreactive Hu–immunolabeled newborn neurons (yellow arrow), along with neurons that are not immunoreactive against pH3 (white arrow) and pH3-immunoreactive extraganglionic cells (cyan arrow). C, Left, Representative image of the LM–MP tissue from an adult mouse housed in the barrier facility at BIDMC when stained with directly conjugated antibody against pH3 (red) shows the presence of significant pH3 immunoreactivity in the tissue. The tissue was also coimmunostained with directly conjugated anti-Hu antibody and the region of interest (white box) when magnified shows (right) the presence of Hu-immunolabeled (green) neurons that colabel for pH3. Nuclei are stained with DAPI (blue). D, The representative image of the adult murine small intestinal LM–MP tissue, when stained with antibodies against CC3 (red) and the pan-neuronal marker Hu (green), shows the presence of CC3 immunoreactivity in a subset of Hu-immunoreactive neurons (yellow arrows), while other neurons in the same and other ganglia do not immunolabel for CC3 (green arrow). Nuclei are labeled with DAPI (blue). Scale bar, 10 µm.

Nestin-expressing and Hu-nonexpressing ENPC express cell cycle marker pH3. Orthogonal (merged and color-segregated) views of a 3D confocal microscopy image showing a myenteric ganglion from an adult murine small intestinal tissue, where the tissue is immunolabeled with antibodies against Hu (gray), pH3 (red), and Nestin (green) and is stained with nuclear dye DAPI (blue). Yellow arrow points out a Nestin- and pH3-immunolabeled cell that does not immunolabel for Hu. The presence of pH3 immunoreactivity and the absence of Hu immunoreactivity in this Nestin-immunolabeled cell suggest it is a cycling enteric neural precursor cell. XY, YZ, and XY planes of the orthogonal views are denoted for every merged and panel-segregated image. Scale bar, 3 μm.

Hu-expressing cells that express cell cycle marker pH3 also exhibit Nestin immunoreactivity. Orthogonal (A) color-segregated and (B) color-merged image of a myenteric ganglion from an adult murine small intestinal tissue, where the tissue is immunolabeled with antibodies against Hu (red), pH3 (gray), and Nestin (green) and is stained with nuclear dye DAPI (blue). Dashed white lines depict a multinucleated contiguous Hu-immunolabeled cell, with white arrows showing that two of the three nuclei are immunolabeled with antibodies against pH3. The pH3⁺ Hu⁺ cells (white arrows) also show positive immunostaining for Nestin. XY, YZ, and XY planes of the orthogonal views are denoted for every merged and color-segregated image. Scale bar, 4 μm.

pH3 immunoreactivity in Hu⁺ cells is unaffected by fixation conditions. Immunostaining adult murine small intestinal LM–MP tissues that were fixed with 4% PFA for (A) 10 min, (B) 15 min, and (C) 20 min and subsequently immunostained with anti-Hu antibodies in ANNA1 serum (green) and pH3 (red) and counterstained with DAPI (blue). Merged and color-segregated views are shown for each of the treatments. White arrows show the presence of pH3 immunoreactivity in the DAPI-stained nuclei of Hu-immunoreactive cells in each of the treatments. White square in panel C is magnified in D and viewed in orthogonal views where XY, YZ, and XZ planes are shown. A contiguous nuclear structure (stained with DAPI, gray) containing three nuclear lobes “#, *, +” are observed within dashed white lines. The nuclear lobe “#” is unstained by pH3 (red) and ANNA1 (green) antibodies, lobe “*” is stained by both pH3 and ANNA1, and lobe “+” is stained by ANNA1 but not by pH3. The three panels show color-segregated views depicting an asymmetric cellular structure where parts of the nuclear structure exhibit Hu and/or pH3 immunoreactivity, while another part does not. XY, YZ, and XY planes of the orthogonal views are denoted for every merged and color-segregated image. Scale bars: A–C, 10 μm; D, 2 μm.

Quantifications of pH3-immunoreactive and CC3-immunoreactive Hu⁺ cells in the adult murine gut. A, Quantification of pH3-immunoreactive Hu⁺ cells in the duodenal tissue of age-matched male and female mice show no significant sex bias in their proportions (p > 0.05, Students’ t test). B, Quantification of pH3-immunoreactive Hu⁺ cells in the ileal tissue of age-matched male and female mice show no significant sex bias in their proportions (p > 0.05, Students’ t test). C, Quantification of pH3-immunoreactive Hu⁺ cells in the duodenal and ileal tissue of age-matched mice show no significant differences in their proportions between the two tissue regions (p > 0.05, Students’ t test). D, Distribution of the percentage of pH3-immunoreactive Hu⁺ cells in small intestinal ganglia containing various numbers of Hu⁺ cells. E, Cubic curve fit (black line) for the distribution of CC3-immunoreactive Hu⁺ cells in small intestinal ganglia containing various numbers of Hu⁺ cells.